Human pluripotent stem cells, such as embryonic stem (ES) cells and embryonic germ (EG) cells, were first isolated in culture without fibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblast feeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblott established continuous cultures of human ES and EG cells using mitotically inactivated mouse feeder layers (Reubinoff et al., 2000; Shamblott et al., 1998; Thomson et al., 1998).
Human ES and EG cells (hESCs) offer unique opportunities for investigating early stages of human development as well as for therapeutic intervention in several disease states, such as liver disease and others. The use of hepatocyte cells, as well as liver precursor cells derived from hESCs for therapeutic liver regeneration, would offer a vast improvement over current cell therapy procedures that utilize cells from donor livers for the treatment of liver disease. Further, the ability to derive hepatocyte cells and liver precursor cells would be beneficial in providing cells for investigating properties of drugs, such as ADME/tox (absorption, distribution, metabolism, excretion/toxicity) studies. However, presently it is not known how to generate liver precursor cells and differentiated liver cells (e.g., mature hepatocyte cells) from hESCs. As such, current cell therapy treatments for liver disease, which utilize liver cells from donor livers, are limited by the scarcity of high quality liver cells needed for transplant. As few as 5 percent of the organs needed for transplant in the United States alone ever become available to a recipient (Evans, et al., (1992). J. Am. Med Assoc., 267:239-246). For example, the American Liver Foundation reports that there are fewer than 3,000 donors for the nearly 30,000 patients who die each year from liver failure. Human embryonic stem cells offer a source of starting material from which to develop substantial quantities of high quality differentiated cells for drug studies as well as human cell therapies.
Two properties that make hESCs uniquely suited to cell therapy applications are pluripoietence and the ability to maintain these cells in culture for prolonged periods. Pluripoietency is defined by the ability of hESCs to differentiate to derivatives of all 3 primary germ layers (endoderm, mesoderm, ectoderm) which, in turn, form all somatic cell types of the mature organism in addition to extraembryonic tissues (e.g. placenta) and germ cells. Although pluripoietency imparts extraordinary utility upon hESCs, this property also poses unique challenges for the study and manipulation of these cells and their derivatives. Owing to the large variety of cell types that may arise in differentiating hESC cultures, the vast majority of cell types are produced at very low efficiencies. Additionally, success in evaluating production of any given cell type depends critically on defining appropriate markers. Achieving efficient, directed differentiation is of great importance for therapeutic application of hESCs.
In order to use hESCs as a starting material to generate cells that are useful in cell therapy applications, it would be advantageous to overcome the foregoing problems. For example, it would be useful to identify and isolate cell types, such as liver precursor cells that can later differentiate into liver cells, as well as other useful cell types.
In addition to efficient direction of the differentiation process, it would also be beneficial to isolate and characterize intermediate cell types along the differentiation pathway towards the hepatocyte cell lineage and to use such cells as appropriate lineage precursors for further steps in the differentiation to mature liver cells.